The virus removal or inactivation capacity of a biopharmaceutical manufacturing process should be proven in a virus validation study. Virus spike concentration and purity can potentially affect the LRV obtained. Additionally, such a study can affect the economy of processing. In particular, if the effect of a more concentrated biotherapeutic on virus-validation profile is minimal, the economy of the filtration can increase. A more robust method can affect economy by maintaining reproducibility despite process variation. The effects of varying IgG concentration, PPV purity and PPV percentage of spike on the performance of Planova 20N virus-removal filters were studied. Design of virus clearance studies, as well as safe and economic virus-filter usage, is considered.

Materials and Methods

Figure 1

Serum or serum-free PPV supernatants were collected, centrifuged at low speed, and 0.45 μm filtered to obtain stock solutions. For purified PPV, following 0.45 μm filtration, ultracentrifugation and density gradient centrifugation were applied. Serum, serum-free, and purified PPV solutions were then used for spiking. The spike percentages were 0.5 or 3.0 vol% for serum-free PPV, 1.0 vol% for serum PPV, and 0.01 vol% for purified PPV. The concentrations of human IgG were 1–30 mg/mL. The non-spiking or spiked IgG solution were prefiltered by Planova 35N to remove aggregates. Filtration was performed in dead-end mode at 0.8 kgf/cm2 (11.4psi).

Results

The Planova 20N flux was not affected by the PPV purity and was identical in the presence and absence of PPV (data not shown). Also, the PPV LRV remained >4 over the filtration volume of 200L/m2 under various spiking conditions. In the non-serum PPV spiking (3vol%) case, a high PPV LRV was maintained past 500L /m2 . (Figure 1) Additionally, for all samples tested, the filtration capacity was not affected by virus purity. (Figure 2)

Discussion

An inaccurate or poorly designed scale-down model can introduce factors that produce incorrect or uninterpretable results in virus-clearance studies. A higher virus spike percentage can overcome virus-assay limitations; however, this can decrease the filtration performance if not chosen wisely. We routinely recommend PPV spike percentages at contract laboratories up to 1% without problem, and XMuLv spike percentages up to 0.5%. Our data shows that purified virus is not a factor under the conditions studied.

Figure 2

Production economy is dictated by the capacities of the materials used to process biotherapeutic material. In particular, a material that consistently performs over a wide variation in challenge can be said to be robust. In the specific case of a virus-removal filter, a virus validation can dictate the V/m2 ratio, based on the margin at which a minimal LRV=4 is maintained. Ideally, a robust virus-removal filter will maintain LRV=4 through a wide concentration range. In the case of MAb's, this range could be 1–5mg/mL to upwards of 20mg/mL, or higher.